Application of a Fluorescent Probe in Detecting Sulfur Dioxide Derivatives

Abstract

The present invention provides an application of a fluorescent probe for detecting sulfur dioxide derivatives. The chemical structural formula of the fluorescent probe is, where R is an alkyl group. When detecting sulfur dioxide derivatives in cells, the fluorescent probe of the present invention does not require a washing process, has simple operation, and can reduce damage to cells, and has the advantages of simple use and high application value.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent probes, and particularly relates to an application of a fluorescent probe for detecting sulfur dioxide derivatives. Background Art

[0002] Sulfur dioxide (SO 2 ) plays an important role in the environment and organisms: Epidemiological studies suggest that excessive intake of sulfur dioxide and its derivatives may cause respiratory diseases such as chronic bronchitis, asthma, and emphysema, as well as neurological disorders such as stroke and migraine. In severe cases, it may even cause lung cancer. Sulfur dioxide mainly exists in aqueous solution in the forms of sulfite (SO 3 2- ) and bisulfite (HSO 3 2- ). Such compounds have antibacterial and antioxidant effects. Therefore, they are commonly used as preservatives and antioxidants in food processing, pharmaceutical preparation, and wine-making processes. In organisms, an appropriate amount of sulfur dioxide helps regulate the dilation of blood vessels in the body, and sulfur dioxide derivatives can be produced in organisms. However, the specific mechanism of sulfur dioxide and its derivatives in organisms is not yet clear. Therefore, developing a method that can efficiently detect sulfur dioxide derivatives in organisms is of great significance for medical diagnosis and disease research.

[0003] Currently, there are many methods for detecting sulfur dioxide, such as electrochemical method, capillary electrophoresis method, high-performance liquid chromatography method, and spectrophotometry. However, these detection methods require complex operation procedures and damage the samples. Therefore, it is difficult to achieve non-invasive, highly sensitive, and real-time detection of changes in sulfur dioxide in organisms. Recently, organic fluorescent probes based on fluorescence imaging technology have become a powerful technique, which can in-situ, non-destructively, and visually detect changes in sulfur dioxide derivatives. At the same time, this method causes less damage to the samples. Currently, many probes for detecting sulfur dioxide derivatives have been designed. However, these probes all require a washing process, which will cause certain damage to cells. Therefore, there is an urgent need for a wash-free fluorescent probe that can observe sulfur dioxide derivatives in cells. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of a fluorescent probe for detecting sulfur dioxide derivatives in view of the deficiencies of the prior art.

[0005] The technical solution of the present invention is as follows:

[0006] An application of a fluorescent probe for detecting sulfur dioxide derivatives, the chemical structural formula of the fluorescent probe is , where R is an alkyl group. This fluorescent probe consists of two parts: pyrrole and benzindole iodide. The double bonds of these two parts are easily combined with SO 2Derivative reaction, which is the reason for its response to sulfur dioxide derivatives.

[0007] As a further improvement of the technical solution, R is an alkyl group with 1 - 3 carbon atoms.

[0008] As a further improvement of the technical solution, R is a methyl group.

[0009] As a further improvement of the technical solution, the fluorescent probe stains cells without washing the cells to observe sulfur dioxide derivatives inside the cells.

[0010] As a further improvement of the technical solution, in order to balance the toxicity of the probe to cells and the staining effect, the concentration of the fluorescent probe for staining cells is 1 - 15 μM.

[0011] As a further improvement of the technical solution, the fluorescent probe is used to detect sulfur dioxide derivatives in a solution.

[0012] As a further improvement of the technical solution, in order to balance the cost and the staining effect, the concentration of the fluorescent probe in the solution is 1 - 15 μM.

[0013] The preparation method of the fluorescent probe is that 2,3,3 - trimethyl - 3H - indole reacts with alkyl iodide to obtain compound (II); then compound (II) and compound (III) are synthesized into the fluorescent probe through the Knoevenagel reaction, and the reaction route is: .

[0014] The present invention has outstanding substantive features and remarkable progress compared with the prior art. Specifically, when the fluorescent probe of the present invention detects sulfur dioxide derivatives inside cells, there is no need for a washing process, which can reduce damage to cells. In addition, the probe has low toxicity and good biocompatibility, which makes it have important application value. Description of the Drawings

[0015] Figure 1 Absorption spectra and fluorescence spectra of PVII (10 μM) in different solvents.

[0016] Figure 2 For the fluorescence spectra of PVII (10 μM) in different concentrations of NaHSO 3 solution, the arrow indicates that the fluorescence intensity decreases with the increase of the concentration of NaHSO 3 concentration.

[0017] Figure 3 Survival results of HeLa cells incubated with different concentrations of PVII.

[0018] Figure 4 For PVII stained with NaHSO 3Confocal fluorescence images of HeLa cells at different times. Detailed implementation manners

[0019] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings in the detailed implementation manners. In the examples, the materials, reagents, etc. used are obtained from commercial sources unless otherwise specified.

[0020] In the examples, the materials, reagents, etc. used are obtained from commercial sources unless otherwise specified.

[0021] Example 1

[0022] Synthesis of (E)-1,3,3-trimethyl-2-(2-(1-methyl-1H-pyrrol)vinyl)-3H-indol-1-ium iodide (abbreviated as PVII)

[0023] The reaction route is as follows:

[0024]

[0025] 1) Synthesis of benzindole iodide (Compound 2)

[0026] Compound 1 (2,3,3-trimethyl-3H-indole, 1.98 mL, 10 mmol) and methyl iodide (1.72 mL, 10 mmol) were dissolved in 20 mL of anhydrous ethanol, and stirred in a flask at room temperature for 1 hour. Then refluxed at 100 °C for 8 h, cooled and filtered, and washed 3 times with anhydrous EtOH. After drying, a white solid, namely Compound 2 (mass: 3.23 g, yield: 90%), was obtained. 1 H NMR (300 MHz, DMSO- d 6 ), δ (ppm): 7.89 - 7.94 (m, 1H), 7.80 - 7.86 (m, 1H), 7.58 - 7.67 (m, 2H), 3.97 (s, 3H), 2.78 (s, 3H), 1.53 (s, 6H).

[0027] 2) Synthesis of the probe PVII

[0028] Compound 2 (0.301 g, 1 mmol) and Compound 3 (0.109 g, 1 mmol) were dissolved in 20 mL of methanol, stirred in a flask for 1 h, and 5 drops of piperidine were added. After stirring, refluxed at 85 °C for 8 h, cooled to room temperature, and washed with petroleum ether. Using CH 2 Cl 2 / CH 3The OH mixture (10:1 - 6:1, v / v) was used as the eluent for column chromatography purification to obtain the yellow solid, which is the probe PVII (mass: 0.25 g, yield: 62%). 1 H NMR (400MHz, DMSO- d 6 ) δ (ppm): 8.02 (d, J=16.0 Hz, 1H), 7.75 (d, J=24.0 Hz, 1H), 7.68 (q, J=2.6 Hz, 1H), 7.55-7.59 (m,2H), 7.50 (t, J=5.3 Hz, 1H), 7.19 (d, J=16.0 Hz, 1H), 6.49 (q, J=2.6 Hz, 1H),7.68 (q, J=2.6 Hz, 1H), 3.96 (d, J=8.0 Hz, 6H), 1.75 (s, 6H). 13 C NMR (400MHz,DMSO-d 6 ), δ (ppm):180.14, 143.01, 142.54, 139.82, 136.16, 132.13, 128.20,120.49, 114.21, 113.36, 105.76, 51.35, 34.73, 33.70, 26.97. HRMS (m / z):calculated 265.17; found: 265.32. C 18 H 21 IN 2 。

[0029] Example 2 Photophysical Property Test Experiment

[0030] Test solutions containing 10 μM PVII were prepared with different solvents, and the absorption spectra and fluorescence emission spectra of the above solutions were measured using a UV-Vis spectrophotometer and a fluorescence spectrometer, respectively, as shown in A and B of Figure 1 . Different concentrations of NaHSO 3 were added to the above solutions, and the fluorescence emission spectra were measured using a fluorescence spectrometer. The results are shown in Figure 2 .

[0031] In Figure 1 A, the probe PVII has an absorption peak at 470 nm, and the absorption peak range is 380 - 540 nm. From Figure 1As can be seen from B, the probe has a fluorescence peak in the range of 480 - 600 nm. The fluorescence intensity of the probe is the strongest in glycerol and weaker in other low-viscosity solvents. This property is beneficial for its high-fidelity imaging inside cells under cell-washing-free conditions. In Figure 2 as the concentration of NaHSO 3 increases, the fluorescence intensity decreases. This indicates that the probe responds significantly to sulfur dioxide derivatives.

[0032] Example 3 Toxicity Test of Probe PVII

[0033] The cytotoxicity of living cells was determined by the standard MTT method. HeLa cells in the logarithmic growth phase were seeded in 96-well plates (about 1×10 4 cells / well), and the wells were filled with cell-free medium as the blank group. The seeded cells were placed in an incubator at 37°C and 5% CO 2 for 24 h, and then PVII at concentrations of 0, 2, 5, 10, and 15 μM was added to the wells as the experimental groups. In addition, DMEM culture medium containing 0.2% DMSO at the final concentration was added as the control group. The cells were incubated at 37°C and 5% CO 2 for 18 hours. Then, MTT (5 mg / mL) was added to each well, and after incubation at 37°C for 4 h, 100 μL of DMSO was added. After incubation for another 20 minutes, the absorbance of each well at 490 nm was measured using a microplate reader, and the cytotoxicity experiment was repeated 4 times.

[0034] The cell survival rate can be calculated by the following formula:

[0035]

[0036] where A sample is the absorbance of the experimental group, A c is the absorbance of the control group, and A b is the absorbance of the blank group.

[0037] The results are shown in Figure 3 , and the results show that after incubating HeLa cells with 15 μM of PVII for 18 hours, the cell survival rate is still as high as 78%, indicating that the toxicity of the probe is very low.

[0038] Example 4 Co-localization Experiment of Probe PVII in Living Cells

[0039] HeLa cells were cultured adherently in high-glucose culture medium containing 10% fetal bovine serum at 37°C and 5% CO 2Cultivate in a saturated humidity incubator, change the culture medium every 2 - 3 days, and perform subculture. When the cells grow to the logarithmic phase, perform slide culture: ① Immerse the cover glass in absolute ethanol for 30 min, dry it with an alcohol lamp and put it into a disposable 35 mm culture dish for standby; ② Wash the cells grown in a 100 mL cell bottle three times with PBS, digest with 1 mL of 0.25% trypsin for 3 - 5 minutes, carefully pour out the trypsin, add fresh culture medium and pipette evenly and count the cells. Control the cell density by the addition amount of the culture medium to make the final cell concentration 1×10 5 cells per milliliter, then inoculate into the above-mentioned culture dish containing the cover glass, and place it in a 5% CO 2 incubator for culture to make the cells adhere tightly to the culture dish. After the HeLa cells grow on the slide and cover the cover glass, they are used for cell experiments.

[0040] Prepare a probe stock solution with a concentration of 1 mM using DMSO. Incubate the cultured active HeLa cells in a culture medium containing 5 μM PVII for 15 min, add 2 mM of NaHSO 3 solution, and observe with a laser confocal microscope. Record the colored parts in the cells, fluorescence distribution and brightness changes, co-localization information, etc. The results are shown in Figure 4 .

[0041] Figure 4 In [figure number], A is the confocal microscope image of active HeLa cells stained with probe PVII (5 μM, 15 min) and treated with 2 mM of NaHSO 3 solution for different times. Figure 4 In [figure number], B is the relative fluorescence intensity of active HeLa cells at different times. Among them, the excitation wavelength of PVII in the green light channel is 488 nm, and the fluorescence collection wavelength is 500 - 600 nm. As time prolongs, the fluorescence intensity of the probe decreases. This confirms that the probe can observe the changes of exogenous sulfur dioxide derivatives inside and outside the cells.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that it is still possible to modify the specific implementation manners of the present invention or perform equivalent replacements for some technical features; without departing from the spirit of the technical solutions of the present invention, they should all be covered within the scope of the technical solutions claimed by the present invention.

Claims

1. Application of a fluorescent probe for detecting sulfur dioxide derivatives, Characterized in that, The chemical structural formula of the fluorescent probe is , where R is an alkyl group.

2. The application according to claim 1, Characterized in that, R is an alkyl group having 1 to 3 carbon atoms.

3. The application according to claim 2, Characterized in that, R is methyl.

4. The application according to any one of claims 1 to 3, Characterized in that, After the cells are stained with the fluorescent probe, the cells do not need to be washed, and the sulfur dioxide derivatives in the cells can be directly observed.

5. The application according to claim 4, Characterized in that, The concentration of the fluorescent probe for staining cells is 1 to 15 μM.

6. The application according to any one of claims 1 to 3, Characterized in that, The fluorescent probe is used to detect sulfur dioxide derivatives in a solution.

7. The application according to claim 6, Characterized in that, The concentration of the fluorescent probe in the solution is 1 to 15 μM.

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